During the proposed grant period I will study the following aspects of the process of RNA-dependent DNA synthesis by RNA tumor viruses. (l) Structural and functional aspects of DNA polymerase from avian RNA tumor viruses. I propose to isolate the small and the large subunits of purified DNA polymerase of avian myeloblastosis virus (AMV) and Rous sarcoma virus (RSV). I will investigate the role of the large subunit in the process of synthesis of DNA and examine the possibility that the large subunit is a host protein. I plan to use peptide mapping to localize the lesion in the DNA polymerase of temperature sensitive mutants of RSV. The DNA polymerases isolated from revertants of these mutants will also be characterized. (2) Mechanism of synthesis of double-stranded DNA by AMV DNA polymerase. I will carry out alpha-P32 transfer experiments to test whether an RNA primer is required to initiate the synthesis of the second strand of DNA. Gamma-P32-labelled deoxyribonucleoside triphosphates will be used as substrates to determine whether the second strand of DNA is synthesized de novo. (3) Properties of murine leukemia virus (MLV) DNA polymerase. Purified DNA polymerase from MLV is unable to utilize 60-70S viral RNA on a template in vitro. I plan to test whether the addition of viral or host cell factors allows 60-70S RNA to be used as a template. I also plan to study the effect of ribonuclease H on the process of RNA-dependent DNA synthesis.